Journal: The Journal of Neuroscience

Article Title: ER-X: A Novel, Plasma Membrane-Associated, Putative Estrogen Receptor That Is Regulated during Development and after Ischemic Brain Injury

doi: 10.1523/JNEUROSCI.22-19-08391.2002

Figure Lengend Snippet: ER-X is neither ER-α nor ER-β.a, Western immunoblots of P7 wild-type and ERKO neocortex and adult wild-type mouse ovary, using antibodies to the LBDs of ER-α (Santa Cruz Biotechnology; MC-20; ovary and neocortex) and ER-β (Zymed; ovary). The apparent MW of ER-X (∼62–63 kDa) is clearly different from the MW of the mouse ER-α (67 kDa) and ER-β (60 kDa) ovarian controls. b, Whereas P7 wild-type neocortex contained both the 67 kDa ER-α and the ∼62–63 kDa ER-X bands, P7 ERKO tissues expressed only the ∼62–63 kDa ER-X band. P7 wild-type and ERKO neocortical CLM preparations were greatly enriched with the ∼62–63 kDa protein. A striking reversal of the ER-α/ER-X ratio was seen in wild-type CLM preparations, in which the ∼62–63 kDa form was highly enriched, whereas authentic 67 kDa ER-α was considerably diminished. c, Absence of ER-β from the plasma membrane, CLM, and non-CLM regions. Note the total absence of ER-β from the wild-type plasma membrane and the CLM and non-CLM fractions. Note also the nuclear concentration of the 60 and 64 kDa isoforms of ER-β. PM, Plasma membrane;non-CLM, non-caveolar-like membrane; CLM, caveolar-like membrane.

Article Snippet: Electrophoretically separated CLMs on PVDF membranes were probed with antibodies specific for ER-α [C1355, (Upstate Biotechnology); MC20 (Santa Cruz Biotechnology)]; ER-β (Zymed); flotillin (Transduction Labs) and other caveolar-resident proteins: e.g., PKC-α and PKC-γ (Transduction Labs) and noncaveolar-resident proteins: e.g., paxillin (Transduction Labs)].

Techniques: Western Blot, Clinical Proteomics, Membrane, Concentration Assay

Journal: The Journal of Neuroscience

Article Title: ER-X: A Novel, Plasma Membrane-Associated, Putative Estrogen Receptor That Is Regulated during Development and after Ischemic Brain Injury

doi: 10.1523/JNEUROSCI.22-19-08391.2002

Figure Lengend Snippet: Estrogen-induced activation of ERK1/2 in CLMs and PNS. Western immunoblots: a, exposure of highly purified, P7 ERKO neocortical CLMs to 17α-estradiol (0.1 nm) and 17β-estradiol (10 nm) for 30 min elicited MEK-dependent (U0126) phosphorylation of ERK1 and ERK2. Non-CLM regions were unresponsive. Densitometry confirmed equal loading of protein. b, Exposure of P7 wild-type neocortical PNS to 17α-estradiol (0.1 nm) and 17β-estradiol (10 nm) for 10 min, 37°C elicited MEK-dependent (U0126) phosphorylation of ERK1 and ERK2. Note that, not only did the ER-α-selective ligand PPT reduce ERK phosphorylation levels below baseline (0) very significantly, but that the level of ERK1/2 phosphorylation, elicited by 17β-estradiol, was also significantly lower than after exposure to 17α-estradiol. This difference may be attributed to the fact that P7 wild-type neocortex is also enriched in ER-α which, because it is activated by 17β- (but not 17-α) estradiol and exerts its inhibitory effect on ERK1/2, as was also seen after exposure to PPT. Bottom blots,Reprobing with antibodies to nonphosphorylated ERK1/2 to verify equal loading of ERK protein across lanes. Densitometry confirmed equal loading. c, Densitometric analysis of ERK activation in wild-type PNS shown in b. These findings confirm that ER-α is a strong inhibitor of ERK activation, a measure of which is shown by the ability of PPT to effectively prevent ERK activation even in the face of the strong activation of ERK elicited by the PPT vehicle ethanol.

Article Snippet: Electrophoretically separated CLMs on PVDF membranes were probed with antibodies specific for ER-α [C1355, (Upstate Biotechnology); MC20 (Santa Cruz Biotechnology)]; ER-β (Zymed); flotillin (Transduction Labs) and other caveolar-resident proteins: e.g., PKC-α and PKC-γ (Transduction Labs) and noncaveolar-resident proteins: e.g., paxillin (Transduction Labs)].

Techniques: Activation Assay, Western Blot, Purification, Phospho-proteomics

Journal: The Journal of Neuroscience

Article Title: ER-X: A Novel, Plasma Membrane-Associated, Putative Estrogen Receptor That Is Regulated during Development and after Ischemic Brain Injury

doi: 10.1523/JNEUROSCI.22-19-08391.2002

Figure Lengend Snippet: ER-X is exquisitely sensitive to picomolar concentrations of 17α- and 17β-estradiol. Western immunoblot of ERK1/2 phosphorylation elicited in wild-type neocortical explants by 17β-estradiol (a) and 17α-estradiol (b). Bottom blots, Reprobing with antibodies to total nonphosphorylated ERK1/2 to verify equal loading of ERK1/2 protein across lanes. pERK, phosphoERK. Densitometry confirmed equal loading. Note that significantly higher levels of 17β-estradiol were required for ERK activation, perhaps reflecting the need in wild-type cultures to overcome the inhibitory effect of ER-α on ERK phosphorylation, which, unlike 17α-estradiol, 17β-estradiol activates as well.

Article Snippet: Electrophoretically separated CLMs on PVDF membranes were probed with antibodies specific for ER-α [C1355, (Upstate Biotechnology); MC20 (Santa Cruz Biotechnology)]; ER-β (Zymed); flotillin (Transduction Labs) and other caveolar-resident proteins: e.g., PKC-α and PKC-γ (Transduction Labs) and noncaveolar-resident proteins: e.g., paxillin (Transduction Labs)].

Techniques: Western Blot, Phospho-proteomics, Activation Assay

Journal: The Journal of Neuroscience

Article Title: ER-X: A Novel, Plasma Membrane-Associated, Putative Estrogen Receptor That Is Regulated during Development and after Ischemic Brain Injury

doi: 10.1523/JNEUROSCI.22-19-08391.2002

Figure Lengend Snippet: ER-X has homology with the LBD of ER-α. Whole-mount of a P2 ERKO neocortical explant, 17 d in vitro. The culture was stained for ER-α mRNA by in situ hybridization with a 48 base oligonucleotide probe to an α-specific region of the ER-α LBD (BER2; Miranda and Toran-Allerand, 1992) and shows the ER-α-like mRNA hybridization signal in neocortical neurons. Residual, untranslated ER-α mRNA? A splice variant of ER-α mRNA? Or the mRNA for a novel ER, ER-X?

Article Snippet: Electrophoretically separated CLMs on PVDF membranes were probed with antibodies specific for ER-α [C1355, (Upstate Biotechnology); MC20 (Santa Cruz Biotechnology)]; ER-β (Zymed); flotillin (Transduction Labs) and other caveolar-resident proteins: e.g., PKC-α and PKC-γ (Transduction Labs) and noncaveolar-resident proteins: e.g., paxillin (Transduction Labs)].

Techniques: In Vitro, Staining, In Situ Hybridization, Hybridization, Variant Assay

Journal: The Journal of Neuroscience

Article Title: ER-X: A Novel, Plasma Membrane-Associated, Putative Estrogen Receptor That Is Regulated during Development and after Ischemic Brain Injury

doi: 10.1523/JNEUROSCI.22-19-08391.2002

Figure Lengend Snippet: Direct evidence in ERKO that ER-X is a neuronal plasma membrane-associated receptor with some homology to the ER-α LBD. A, Using antibodies highly specific for an α-specific region of the LBD of ER-α (C1355), large numbers of immature immunoreactive neocortical ERKO neurons with unstained nuclei are seen. B, The immunoreactivity is clearly localized to the cell membrane and cytoplasm and not in the nucleus.D, E, Antibodies, raised against the full-length ER-α molecule, said to recognize epitopes in the 5′, N-terminal region (6F11), but which we have found also to cross-react significantly with ER-β, show widespread nuclear labeling with no cytoplasmic or membrane labeling seen. The nuclear labeling observed most likely reflects intranuclear ER-β, which is normally expressed in both wild-type and ERKO neocortical neurons. C, CLM association of ER-X in ERKO neocortical neurons was further documented at the ultrastructural level by demonstrating immunoreactive flotillin (gold particles), colocalized with immunoreactivity for the ER-α LBD(horseradish peroxidase) on a mushroom-like neocortical dendritic spine. Scale bars, 10 μm.

Article Snippet: Electrophoretically separated CLMs on PVDF membranes were probed with antibodies specific for ER-α [C1355, (Upstate Biotechnology); MC20 (Santa Cruz Biotechnology)]; ER-β (Zymed); flotillin (Transduction Labs) and other caveolar-resident proteins: e.g., PKC-α and PKC-γ (Transduction Labs) and noncaveolar-resident proteins: e.g., paxillin (Transduction Labs)].

Techniques: Clinical Proteomics, Membrane, Labeling

Journal: The Journal of Neuroscience

Article Title: ER-X: A Novel, Plasma Membrane-Associated, Putative Estrogen Receptor That Is Regulated during Development and after Ischemic Brain Injury

doi: 10.1523/JNEUROSCI.22-19-08391.2002

Figure Lengend Snippet: ER-X is upregulated after ischemic brain injury in the adult. Comparison of ER-α and ER-X expression in the infarcted and noninfarcted adult neocortex. After a large ischemic infarct in the neocortex produced by middle cerebral artery occlusion, there was not only upregulation of ER-α expression in the penumbra of the ligated, ischemic side but also upregulation of ER-X therein as well, suggesting re-expression of a developmental mechanism normally latent in the adult. Note the lack of significant ER-X expression on the noninfarcted side. MCF-7 mammary tumor cells and adult uterus = ER-α controls; P7 neocortex = ER-X control.

Article Snippet: Electrophoretically separated CLMs on PVDF membranes were probed with antibodies specific for ER-α [C1355, (Upstate Biotechnology); MC20 (Santa Cruz Biotechnology)]; ER-β (Zymed); flotillin (Transduction Labs) and other caveolar-resident proteins: e.g., PKC-α and PKC-γ (Transduction Labs) and noncaveolar-resident proteins: e.g., paxillin (Transduction Labs)].

Techniques: Comparison, Expressing, Produced, Control

Journal: The Journal of Physiological Sciences : JPS

Article Title: 17β-Estradiol-induced enhancement of estrogen receptor biosynthesis via MAPK pathway in mouse skeletal muscle myoblasts

doi: 10.1007/s12576-009-0023-0

Figure Lengend Snippet: Distribution of estrogen receptors in various organs and tissues of adult mouse. a Western blot analysis of whole-cell fractions with a specific monoclonal estrogen receptor (ER) antibody. Upper panel immunoreactive bands corresponding to the ER protein (66 kDa). The histogram (lower) indicates the results of densitometric analysis for a 66-kDa band (mean ± SE, n = 4). Relative expression level with respect to the ovary is shown in %. From left to right: skeletal muscle, uterus, lung, adipose tissue, kidney, and myocardium. b Relative expression (to ovary) of whole cell and nonnuclear ER proteins obtained from C2C12 mouse myoblasts (n = 3)

Article Snippet: The blotted PVDF membrane was successively incubated with primary monoclonal anti-ERα mouse antibody (Upstate Biotechnology, Lake Placid, NY), and, after fpur washes, in 0.05% Tween 20-TBS (20 mM Tris-HCl, 150 mM NaCl) for 5 min, with secondary antibodies conjugated to horseradish peroxidase (Amersham Biosciences, Piscataway, NJ) (for each diluted 1,000 times; 1 h at room temperature).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: Structural evolution of the estrogen receptor regulatory domain

doi: 10.1101/2024.12.18.629305

Figure Lengend Snippet: a , Domain architecture of full-length ERs (NTD: N-terminal domain; AF1: activation-function 1; DBD: DNA-binding domain; LBD: ligand-binding domain; AF2: activation-function 2). b , Maximum-likelihood phylogenetic tree of 1,051 ER LBD sequences from vertebrates and protostomes; the branch corresponding to protostomes is compressed. c , X-ray crystal structure of the rf ERγ LBD solved at 1.95 Å resolution revealing conservation of protein folding, homodimer assembly, AF2 conformation and d E2 binding mode. Secondary structure elements are labelled in bold and residues are labelled accordingly (using h ERα LBD numbering for consistency). In d , residues that are conserved with ERα and ERβ homologs from human and rainbowfish are coloured in black. Sidechains and E2 are shown as sticks, and the simulated annealing composite omit 2F o -F c electron density map (1.5 σ) is shown as blue mesh. e , Representative snapshots of the F425 χ 1 sidechain dihedral angle obtained from K-means clustering (k=10) of triplicate 1 µs all-atom molecular dynamics (MD) simulations. Residue sidechains and E2 are shown as sticks (E2: yellow; F/M/I421: orange; F425: green; I/L429: purple). f , Histogram showing aggregate F425 χ 1 sidechain dihedral angle populations for each homolog. g , Top-down perspective of the rf ERγ LBD homodimer showing structural plasticity of the dimerisation interface; notable residues are shown as sticks and coloured according to the legend below. h , Hydrophobic surface representation of one subunit from the rf ERγ LBD homodimer and opposing residue sidechains.

Article Snippet: Codon-optimised rf ERα, rf ERβ and rf ERγ LBD primary sequences were cloned into pET-21b ( Twist Bioscience ); h ERα and h ERβ LBD primary sequences were cloned into pET-11a vector ( Genscript ).

Techniques: Activation Assay, Binding Assay, Ligand Binding Assay, Residue

Journal: bioRxiv

Article Title: Structural evolution of the estrogen receptor regulatory domain

doi: 10.1101/2024.12.18.629305

Figure Lengend Snippet: a, An alignment comparing the primary amino acid sequences of each homolog. The numbering at sequence ends are the start and finish residue positions of each LBD; however, herein h ERα LBD numbering is used for consistency. Residue positions encoding the ligand binding pocket (LBP) are shown in green; the activation function-2 interface (AF2) in orange; and the dimerisation interface shown in purple. b, X-ray crystal structures of the h ERα LBD bound to estradiol (E2) and h SRC1 coactivator peptide (PDB: 3UUD); the rf ERα LBD bound to E2 and h SRC2 coactivator peptide (PDB: 9D8Q); the h ERβ LBD (PDB: 3OLL) bound to E2 and h SRC1 coactivator peptide; a computational homology model of the rf ERβ LBD bound to E2; and X-ray crystal structure of the rf ERγ LBD (PDB: 9D8R) bound to E2. Each structure is shown as the homodimer, with the second subunit coloured in grey. c, Close-up perspective of the LBP showing the conservation of E2 binding mode across homologs. d, The binding mode of coactivator peptides solved with h ERα, rf ERα and h ERβ, and all peptides modelled onto the AF2 of rf ERβ and rf ERγ LBDs, revealing conservation of structure and LxxLL motif recognition.

Article Snippet: Codon-optimised rf ERα, rf ERβ and rf ERγ LBD primary sequences were cloned into pET-21b ( Twist Bioscience ); h ERα and h ERβ LBD primary sequences were cloned into pET-11a vector ( Genscript ).

Techniques: Sequencing, Residue, Ligand Binding Assay, Activation Assay, Binding Assay

Journal: bioRxiv

Article Title: Structural evolution of the estrogen receptor regulatory domain

doi: 10.1101/2024.12.18.629305

Figure Lengend Snippet: a, Modelling of all possible rotamer orientations of the F421 substitution in ERα and ERβ backgrounds with the Dunbrack rotamer library shows clashing of the phenyl sidechain with E2 and neighbouring structure, compared to the wildtype amino acid. b, Comparison of the structure and sequence of H6 between homologs. c, Secondary structure analysis of H6 (residues 409 to 417) calculated from a 1 µs all-atom molecular dynamics (MD) simulation using the DSSP algorithm in MDTraj , .

Article Snippet: Codon-optimised rf ERα, rf ERβ and rf ERγ LBD primary sequences were cloned into pET-21b ( Twist Bioscience ); h ERα and h ERβ LBD primary sequences were cloned into pET-11a vector ( Genscript ).

Techniques: Comparison, Sequencing

Journal: bioRxiv

Article Title: Structural evolution of the estrogen receptor regulatory domain

doi: 10.1101/2024.12.18.629305

Figure Lengend Snippet: a , Schematic outlining the approach. See Materials and Methods for a detailed description and breakdown of each step. b , A contact matrix showing all non-covalent residue interactions between residues (all homologs) coloured by probability. c , Graph of the allosteric network comprised of components 1 and 2 (C1: purple; C2: blue). Node size is proportional to the betweenness centrality (or importance) of each node. d , Allosteric network mapped to the crystal structure of the wildtype h ERα LBD (PDB: 1GWR). Residue sidechains are shown as spheres and coloured by component. e , Structural analysis of the allosteric network. Conserved residues shared are labelled in black while non-conserved residues are labelled in red with varying amino acids annotated. f , Same as in c but for the components deemed important for supporting protein folding. g , The folding network mapped to structure. Residue sidechains are shown as spheres and coloured by component (see key). h ) Perspective of the α-helical bundle and folding network components. Residue annotation follows convention of d .

Article Snippet: Codon-optimised rf ERα, rf ERβ and rf ERγ LBD primary sequences were cloned into pET-21b ( Twist Bioscience ); h ERα and h ERβ LBD primary sequences were cloned into pET-11a vector ( Genscript ).

Techniques: Residue

Journal: bioRxiv

Article Title: Structural evolution of the estrogen receptor regulatory domain

doi: 10.1101/2024.12.18.629305

Figure Lengend Snippet: Left: backbone heavy-atom RMSD for each 1 µs replicate; middle: estradiol (E2) RMSD for each 1 µs replicate; right: helix-12 (H12) backbone RMSD for each 1 µs replicate. a-e, in order: h ERα LBD, h ERβ LBD, rf ERα LBD, rf ERβ LBD and rf ERγ LBD. The 5 ns rolling-average (solid line) is superimposed above raw RMSD values.

Article Snippet: Codon-optimised rf ERα, rf ERβ and rf ERγ LBD primary sequences were cloned into pET-21b ( Twist Bioscience ); h ERα and h ERβ LBD primary sequences were cloned into pET-11a vector ( Genscript ).

Techniques:

Journal: bioRxiv

Article Title: Structural evolution of the estrogen receptor regulatory domain

doi: 10.1101/2024.12.18.629305

Figure Lengend Snippet: a , Evaluation of variability within functional regions of 1,051 ER LBD sequences (α, β and γ) from phylogenetically diverse taxa. Area under the curve (AUC) was used as a quantitative measure of constraint, calculated by plotting the fraction of unique genotypes in the alignment encoding functional regions (e.g., the dimerisation interface) against the fraction of the total sequence population. b , The evolutionary conservation of each residue position along the length of the LBD determined by the Jensen-Shannon divergence (JSD). c , Evolutionary conservation of each residue position encoding the ligand binding pocket (LBP), activation function-2 (AF2), allosteric network (AN), dimerisation interface (DI), folding network (FN) and non-functional residues (NF), shown as a box plot. The box plot shows the median and interquartile range (IQR); whiskers represent the distribution of the data as a function of the IQR. Mann-Whitney U-test was used to determine statistical significance compared to NF residues (LBP: P = 0.00095; AF2: P = 0.00303; AN: P = 0.03354; DI: P = 0.12424; FN: P = 0.10295). d , The evolutionary conservation of each residue position mapped to the h ERα LBD (1GWR; missing loops were modelled in ICM-Pro Molsoft for visualisation purposes) highlights the architecture of constraint within the ER LBD. Each residue position comprising regions are shown as spheres (Cα atom) and coloured according to the level of conservation (red = low; blue = high). Refer to for the network analysis.

Article Snippet: Codon-optimised rf ERα, rf ERβ and rf ERγ LBD primary sequences were cloned into pET-21b ( Twist Bioscience ); h ERα and h ERβ LBD primary sequences were cloned into pET-11a vector ( Genscript ).

Techniques: Functional Assay, Sequencing, Residue, Ligand Binding Assay, Activation Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Structural evolution of the estrogen receptor regulatory domain

doi: 10.1101/2024.12.18.629305

Figure Lengend Snippet: a-d, Sequence space networks of the ligand binding pocket (LBP), activation function-2 interface (AF2), allosteric network and dimerisation interface. A genotype is depicted as a node and are connected by an edge to another node if the Hamming distance between them is equal to 1, or 1 residue substitution (i.e., the Hamming distance between AGFST and AFFST is 1). The colour of each node corresponds to the number of times each genotype is observed in the alignment of 1,051 ER LBD sequences. For visual clarity only connected nodes are shown. e, The number of mutational steps that each genotype (a line) must take to visit all other genotypes in the network, given as the Hamming distance, or shortest-mutational paths. The solid orange line represents the mean trajectory.

Article Snippet: Codon-optimised rf ERα, rf ERβ and rf ERγ LBD primary sequences were cloned into pET-21b ( Twist Bioscience ); h ERα and h ERβ LBD primary sequences were cloned into pET-11a vector ( Genscript ).

Techniques: Sequencing, Ligand Binding Assay, Activation Assay, Residue

Journal: bioRxiv

Article Title: Structural evolution of the estrogen receptor regulatory domain

doi: 10.1101/2024.12.18.629305

Figure Lengend Snippet: a ) The burden, or total number, of unique variants within each functional region shown as a grouped barplot (LBP: ligand binding pocket; AF2: activation function-2; AN: allosteric network; DI: dimerisation interface; FN: folding network; NF: non-functional residues). Note that a residue position may harbour more than one unique variant. b , Venn diagram showing the distribution of shared residue positions harbouring variants between h ERα and h ERβ LBDs. c , Histogram showing the distribution of the evolutionary conservation (Jensen-Shannon divergence) of each variant residue position for both h ERα and h ERβ, as well as residue positions that share variant occurrence. d , Average solvent accessible surface area (SASA) of residue positions harbouring variants calculated from the homodimeric crystal structures of the h ERα (PDB: 1GWR) and h ERβ (PDB: 3OLL) LBD. Missing residues in loops were modelled with ICM-Pro Molsoft. e , Average Cα-atom root-mean squared fluctuations (RMSF) of residue positions harbouring variants calculated from the replicate MD simulations. f , Molecular architecture of residue positions harbouring variants mapped to h ERα (PDB: 1GWR) and h ERβ (PDB: 3OLL) LBDs. Residues are shown as spheres (Cα atoms) and coloured according to variant burden. The black box highlights the differences between variant burden in ligand binding pocket and of allosteric network residues between h ERα and h ERβ LBDs. Whiskers in d and e represent median and interquartile range (IQR). g , A structural perspective of the ligand binding pocket and allosteric network residues of the h ERα LBD (left) and h ERβ LBD (right). Important residue sidechains are shown as sticks. Residue positions with variants are identified by a sphere which is coloured to the total burden of unique variants. Wildtype residues are labelled in black, while identified variants are labelled in red to highlight changes. h , Structural comparison between wildtype h ERα (PDB: 1GWR) and Y537S (gain-of-function; PDB: 3UUD). Adjacent allosteric network residues are shown as sticks and the residue at position 537 is coloured teal

Article Snippet: Codon-optimised rf ERα, rf ERβ and rf ERγ LBD primary sequences were cloned into pET-21b ( Twist Bioscience ); h ERα and h ERβ LBD primary sequences were cloned into pET-11a vector ( Genscript ).

Techniques: Functional Assay, Ligand Binding Assay, Activation Assay, Residue, Variant Assay, Solvent, Comparison

Journal: bioRxiv

Article Title: Structural evolution of the estrogen receptor regulatory domain

doi: 10.1101/2024.12.18.629305

Figure Lengend Snippet: a, Burden of unique missense variants across each position of the h ERα and h ERβ LBD. Variants were obtained from gnomAD( 48 ). b, Significance p-values calculated from hypergeometric tests for statistical depletion of variants in each functional region relative to all positions within the LBD. A significance threshold of P = 0.05 was used. Only the ligand binding pocket (LBP) and allosteric network (AN) in the h ERα LBD were significantly depleted of variants, as compared to the activation function-2 interface (AF2), dimerisation interface (DI) and folding network (NF). No functional region was significantly depleted of variants in the h ERβ LBD. c, Structural modelling of example h ERα variants M421V (putative benign) and R394S, (putative loss-of-function) and Y537S (gain-of-function; PDB: 3UUD).

Article Snippet: Codon-optimised rf ERα, rf ERβ and rf ERγ LBD primary sequences were cloned into pET-21b ( Twist Bioscience ); h ERα and h ERβ LBD primary sequences were cloned into pET-11a vector ( Genscript ).

Techniques: Functional Assay, Ligand Binding Assay, Activation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Estrogen receptor ? sustains epithelial differentiation by regulating prolyl hydroxylase 2 transcription

doi: 10.1073/pnas.1221654110

Figure Lengend Snippet: PHD2 expression is regulated by ligand-dependent activation of ERβ in epithelial cells. (A) ERβ ablation induces dedifferentiaton in PNT1a epithelial cells. ERb expression was ablated in PNT1a cells using two independent shRNAs (shERb#1 and shERb#2). These cells and control cells (shGFP) were maintained in either 2D or 3D culture. Control cells exhibit a distinct epithelial phenotype in 2D and a spheroid shape with a distinct polarity in 3D. In contrast, loss of ERβ expression promotes a mesenchymal phenotype in 2D and invasive outgrowths in 3D. (Scale bars, 100 μm.) Morphological changes observed in response to loss of ERβ are accompanied by diminished E-cadherin expression and an increase in mesenchymal markers (N-cadherin, vimentin, and HIF-1α). Immunoblot on the Far Right demonstrates that PNT1a cells lack expression of ERα in comparison with T47D breast carcinoma cells. (B) ERβ ablation suppresses PHD2 expression. ERβ-ablated PNT1a cells were assessed for PHD2 expression by qPCR and immunoblotting. Loss of ERβ expression is accompanied by a marked reduction in PHD2 mRNA and protein compared with the control cells. The reduction of PHD2 expression as a consequence of ERβ ablation is specific because there is no effect on PHD1 and PHD3 mRNA (*P < 0.05). Immunoblot on the Far Right demonstrates that loss of ERβ in LNCaP cells induces HIF-1α. (C) PHD2 expression is induced by an ERβ ligand, 3β-adiol. PNT1a and LNCaP cells were incubated with DMSO (control) or 5 µM 3β-adiol for 2 d. Cells were examined for PHD2 expression by qPCR and immunoblotting. 3β-Adiol enhances PHD2 mRNA and protein expression significantly. In contrast, 3β-adiol had no effect on PHD2 expression in ERβ-ablated PNT1a cells. (D) 3β-Adiol and DPN but not estradiol enhance PHD2 expression. PHTPP attenuates PHD2 expression in either the absence or presence of 3β-adiol. (E) TGF-β–induced EMT suppresses PHD2 expression. PNT1a cells were cultured in the absence or presence of TGF-β (5 ng/mL) for 3 d and harvested for immunoblotting. TGF-β treatment diminishes PHD2 expression significantly with a concomitant decrease in E-cadherin and ERβ.

Article Snippet: Blots were incubated at 4 °C overnight with primary Abs against ERα or ERβ (GeneTex), E-cadherin (Invitrogen), N-cadherin (Invitrogen), vimentin (Dako), HIF-1α (Novus), PHD2 (Abcam), and β-actin (Sigma) and immune complexes were detected using enhanced chemiluminescence (ECL; Pierce).

Techniques: Expressing, Activation Assay, Control, Western Blot, Comparison, Incubation, Cell Culture

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Estrogen receptor ? sustains epithelial differentiation by regulating prolyl hydroxylase 2 transcription

doi: 10.1073/pnas.1221654110

Figure Lengend Snippet: ERβ regulates PHD2 transcription via a unique ERE, located in the 5′ UTR of the PHD2 gene. (A) Schematic of the wild-type and mutant luciferase reporter constructs used. A 927-bp fragment (−2597 bp to −1670 bp), containing two variant EREs (I-ERE1: TATCAgtaTGAAT and I-ERE2: AATCAggcTGACA), was cloned into a pGL3-promoter construct (WT). M1 represents the mutated version of I-ERE1 (TAAAAGTAAAAAT), whereas M2 represents the mutated version of I-ERE2 (AAAAAGGCAAACA). These constructs were used for subsequent experiments. (B) PNT1a cells were transfected with WT, M1, or M2 luciferase constructs. ERβ-ablated cells (shERβ-1 and shERβ-2) were transfected with the WT construct together with Renilla for normalization. Cells were subsequently incubated for 20 h in the absence or presence of 3β-adiol. Note that the relative luciferase activity (RLA) of the M2 construct is significantly less than that of the WT and M1 constructs and comparable to that of the wild-type construct in ERβ-ablated cells in the absence or presence of 3β-adiol (*P < 0.05). However, only the wild-type and M1 constructs exhibit ligand responsiveness (two- to threefold increase in luciferase activity). (C) Ligand-activated ERβ promotes PHD2 expression in PC3-M cells. PC3-M cells, which have an undetectable level of ERβ, were infected with an empty vector or an HA-ERβ expression vector. ERβ expressing cells (lanes 2 and 3) were incubated with DMSO (control), 3β-adiol (5 µM), or DPN (30 nM) for 24 h and harvested for immunoblotting. ERβ expression alone increases PHD2 expression and this effect is enhanced by either 3β-adiol or DPN. (D) Analyses of ERE-luciferase activity in PC3-M cells. PC3-M cells that express an HA-ERβ or an empty vector (control) were transfected with either WT (±3β-adiol), M1, or M2 constructs together with Renilla. Expression of HA-ERβ increases luciferase activity, which is enhanced by 3β-adiol (*P < 0.05). This enhancement is not apparent with M2. (E) EMSA assays to detect ERβ–DNA complex formation in vitro. Nuclear extracts containing ERβ from 3β-adiol–treated PNT1a cells or ERβ-ablated cells were incubated with 32P-labeled I-ERE2 (WT) in the absence or presence of unlabeled competitors. A distinct protein–DNA complex was detected (arrow) in PNT1a cells but not in ERβ-ablated cells (shERβ-1 and shERβ-2) and this complex was displaced by unlabeled I-ERE2 (WT) but not I-ERE2 (mut) confirming its specificity. (F) ChIP analysis of HA-ERβ–expressing PC3-M cells. PC3-M (+HA-ERβ) or PC3-M cells (empty vector) were incubated in the absence or presence of 3β-adiol for 48 h. Either an HA antibody or IgG (control) were used to precipitate the chromatin. Enrichment on the ERE2 locus by HA was normalized to IgG by qPCR. I-ERE2 mRNA levels exhibit a fourfold induction in HA-ERβ–expressing cells and a sixfold induction upon treatment with 3β-adiol. In contrast, no enrichment in I-ERE2 mRNA is apparent in control PC3-M cells (*P < 0.05) under the same conditions.

Article Snippet: Blots were incubated at 4 °C overnight with primary Abs against ERα or ERβ (GeneTex), E-cadherin (Invitrogen), N-cadherin (Invitrogen), vimentin (Dako), HIF-1α (Novus), PHD2 (Abcam), and β-actin (Sigma) and immune complexes were detected using enhanced chemiluminescence (ECL; Pierce).

Techniques: Mutagenesis, Luciferase, Construct, Variant Assay, Clone Assay, Transfection, Incubation, Activity Assay, Expressing, Infection, Plasmid Preparation, Control, Western Blot, In Vitro, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Estrogen receptor ? sustains epithelial differentiation by regulating prolyl hydroxylase 2 transcription

doi: 10.1073/pnas.1221654110

Figure Lengend Snippet: PHD2 sustains epithelial differentiation. (A) PHD2 ablation promotes dedifferentiation. PHD2-ablated cells (shPHD2#1 and shPHD2#2) and control cells (shGFP) were maintained in either 2D or 3D cultures. Note that the morphology of PHD2-ablated cells is similar to that of ERβ-ablated cells in Fig. 1. (Scale bars, 100 μm.) Immunoblot analysis of these cells indicates that loss of PHD2 induces a decrease in E-cadherin expression and an increase in mesenchymal markers. (B) Inhibition of PHD2 activity induces epithelial dedifferentiation. PNT1a cells were incubated for 2 d with DMSO (control) or 100 µM DMOG, a PHD2 inhibitor. DMOG-treated cells exhibit a mesenchymal phenotype with concomitant increase in vimentin and HIF-1α expression compared with controls. (C) PHD2 ablation induces epithelial dedifferentiation and promotes HIF-1α activity in LNCaP cells. PHD2-ablated LNCaP cells (shPHD2#1 and shPHD2#2) exhibit a mesenchymal phenotype compared with control cells. Loss of PHD2 expression also induces HIF-1α and vimentin expression. These cells were transfected with a reporter gene carrying either a wild-type HRE or a mutated HRE. A significant induction in luciferase activity is seen in PHD2-ablated cells compared with control cells with the wild type HRE-reporter gene but not with the mutant HRE reporter (*P < 0.05). (D) Inhibition of PHD2 activity induces dedifferentiation in LNCaP cells. LNCaP cells were incubated with either DMSO (−) or 100 µM DMOG (+) for 2 d. DMOG-treated cells exhibit a mesenchymal morphology and an increase in vimentin and HIF-1α expression compared with control cells.

Article Snippet: Blots were incubated at 4 °C overnight with primary Abs against ERα or ERβ (GeneTex), E-cadherin (Invitrogen), N-cadherin (Invitrogen), vimentin (Dako), HIF-1α (Novus), PHD2 (Abcam), and β-actin (Sigma) and immune complexes were detected using enhanced chemiluminescence (ECL; Pierce).

Techniques: Control, Western Blot, Expressing, Inhibition, Activity Assay, Incubation, Transfection, Luciferase, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Estrogen receptor ? sustains epithelial differentiation by regulating prolyl hydroxylase 2 transcription

doi: 10.1073/pnas.1221654110

Figure Lengend Snippet: PHD2 expression can rescue an epithelial phenotype in dedifferentiated cells. (A) Expression of PHD2 in ERβ-ablated cells induces a mesenchymal to epithelial transition. Two clones of ERβ-ablated LNCaP cells (shERβ#1 and shERβ#2) were infected with a PHD2 expression vector or empty vector (control). Expression of PHD2 promotes a more epithelial morphology with and a decrease in mesenchymal markers (vimentin, N-cadherin, and HIF-1α). (B) Expression of PHD2 in dedifferentiated cells induces epithelial differentiation that is dependent on PHD2 activity. PC3-M cells were infected with a PHD2 expression vector or empty vector (control). Note that expression of PHD2 promotes an epithelial phenotype with an increase in E-cadherin and a reduction of HIF-1α and vimentin. Subsequent treatment of the cells with DMOG (100 µM) for 3 d induces dedifferentiation and an increase in HIF-1α and vimentin. The changes in HIF-1α expression in response to PHD2 and DMOG inhibition are consistent with the activity of an HRE reporter (bar graph). (C) Expression of a HIF-1α mutant that is resistant to hydroxylation by PHD2 promotes dedifferentiation in cells with an epithelial phenotype. LNCaP cells were infected with expression vectors containing either wild-type HIF-1α, a mutant HIF-1α, or an empty vector (control). Expression of the mutant HIF-1α promotes dedifferentiation in contrast to wild-type HIF-1α. These morphologic changes are consistent with the changes in HIF-1α and E-cadherin expression (immunoblot) and the activity of an HRE reporter (bar graph: *P < 0.05). (Scale bars, 100 μm.)

Article Snippet: Blots were incubated at 4 °C overnight with primary Abs against ERα or ERβ (GeneTex), E-cadherin (Invitrogen), N-cadherin (Invitrogen), vimentin (Dako), HIF-1α (Novus), PHD2 (Abcam), and β-actin (Sigma) and immune complexes were detected using enhanced chemiluminescence (ECL; Pierce).

Techniques: Expressing, Clone Assay, Infection, Plasmid Preparation, Control, Activity Assay, Inhibition, Mutagenesis, Western Blot

Journal: Endocrine-Related Cancer

Article Title: GPER-mediated proliferation and estradiol production in breast cancer-associated fibroblasts

doi: 10.1530/ERC-13-0237

Figure Lengend Snippet: GPER is expressed in primary and immortalized breast CAFs. (A) CAFs were identified by immunofluorescent staining with α-SMA and FAP. ERs were detected in primary breast CAFs, immortalized CAFs (CAF-hTERT), and MCF-7 cells as positive controls. Scale bars: 25 μm. (B) ER mRNA expression was evaluated by quantitative real-time RT-PCR in primary CAFs, CAF-hTERT cells, and MCF-7 cells. Gene expression was normalized to GAPDH , and results are shown as fold changes of mRNA levels compared with MCF-7 cells. The data are shown as means± s.d . for three independent experiments.

Article Snippet: The cells were then incubated overnight at 4 °C with primary antibodies targeting α-SMA, FAP, and GPER (Abcam) and ERα and ERβ (ZSGB-BIO, Beijing, China); all dilutions were 1:150.

Techniques: Staining, Expressing, Quantitative RT-PCR, Gene Expression